Raffinose synthase gene, method for producing raffinose, and transgenic plant

ABSTRACT

Raffinose synthase purified from cucumber is allowed to act on sucrose and galactinol. Thus raffinose is efficiently produced. The function of endogenous raffinose synthase is regulated by transforming a plant with a chimeric gene comprising a raffinose synthase gene and a regulatory region expressible in the plant. Thus a plant, in which raffinose family oligosaccharides are decreased, is created.

TECHNICAL FIELD

The present invention relates to a raffinose synthase, a method forraffinose synthesis based on the use of raffinose synthase or acell-free extract containing the raffinose synthase, DNA coding for theraffinose synthase, and methods for its use to produce altered amount ofraffinose family oligosaccharides in transformed plants. Raffinose isutilized in a variety of fields, as a food material having an activityto proliferate Bifidzbacterium, or as a pharmaceutical to be used, forexample, for solutions of organ preservation.

BACKGROUND ART

Raffinose is one of raffinose family oligosaccharides, in whichgalactose is connected to glucosyl group of sucrose via α-1,6 linkage.The raffinose family oligosaccharides include, for example, stachyosecontaining two connected galactose residues, and verbascose containingthree connected galactose residues, in addition to raffinose. Theseoligosaccharides are widely distributed in plants, for example, seeds ofvarious plants such as beans, rapeseed, and cottonseed containing theseoligosaccharides as reserve carbohydrates; plants belonging toCucurbitaceae such as cucumber and melon containing these sugars astransport sugars; and sugar beet (Beta vulgaris) and rosette leaveshaving acquired cold resistance.

The raffinose family oligosaccharides are biosynthesized as follows.UDP-galactose+myo-inositol→galactinol+UDP  (a)galactinol+sucrose→raffinose+myo-inositol  (b)galactinol+raffinose→stachyose+myo-inositol  (c)

The respective reactions are catalyzed by (a) galactinol synthase (GS:EC 2.4.1.123), (b) raffinose synthase (RS: EC 2.4.1.82), and (c)stachyose synthase (STS: EC 2.4.1.67).

At present, raffinose is extracted from sugar beet, and it is separatedand purified in the sucrose purification process. However, since crystalformation of sucrose is deteriorated by raffinose, sugar beet has beensubjected to breeding and improvement with the aim of decreasing theraffinose content. As a result, the raffinose content in sugar beet nowhas a low value of 0.03% to 0.16% (Enzyme Microb. Technol., Vol. 4, May,130-135 (1982)). Therefore, it is not easy to efficiently obtainraffinose from sugar beet having such a low raffinose content.

As described above, raffinose is contained in mature seeds of plantsbelonging to Leguminosae represented by soybean. Mature seed of soybeancontains, as soybean oligosaccharides, sucrose (content: about 5 t),stachyose (content: about 4%), and raffinose (content: about 1%). Thesoybean oligosaccharides are recovered in a fraction obtained bydeproteinizing defatted soybean, and they are utilized, for example, forfunctional food products after concentration. However, raffinoseoccupies a proportion of 10% of the whole oligosaccharides, and henceraffinose exists in a small amount.

On the other hand, a method for enzymatically synthesizing raffinose hasbeen reported (Trends in Glycoscience and Glycotechnology, 7.34, 149-158(1995)). This method comprises the steps of synthesizing galactobiose inaccordance with a condensation reaction catalyzed by α-galactosidase,and transferring galactosyl group to sucrose by using the galactobioseas a galactosyl group donor in accordance with a galactosyl transferreaction to synthesize raffinose. However, in this reaction, 350 g ofgalactobiose is synthesized from 1.9 kg of lactose hydrolysate, and 100g of raffinose is obtained from 190 g of galactobiose and 760 g ofsucrose. Therefore, the yield of produced raffinose is low, and hencethis synthesis method is not efficient.

Besides the foregoing methods, a method is also conceivable in which aplant having a high raffinose content may be bred by means oftransformation for genes for enzymes included in the biosynthesissystem. For example, Kerr et al. have cloned a gene for galactinolsynthase, and transformed rapeseed therewith (WO 93/02196). As a result,the GS activity was increased, however, the content of the raffinosefamily oligosaccharides was unwillingly decreased. It was impossible toachieve the object to enhance the biosynthesis of the raffinose familyoligosaccharides by transforming the galactinol synthase gene.Therefore, there has not been provided a method for increasing thecontent of the raffinose family oligosaccharides in plant.

On the other hand, it is also demanded to decrease the raffinose familyoligosaccharides. As described above, the raffinose familyoligosaccharides are widely distributed over plants including, seeds ofvarious plants such as beans, for example, soybean, rapeseed, andcottonseed containing these oligosaccharides as storage carbohydrates;plants belonging to Cucurbitaceae such as cucumber and melon containingthese oligosaccharides as transport sugars; and sugar beet and rosetteleaves having acquired cold resistance. Meals obtained after extractionof oil, for example, from soybean, rapeseed, and cotton contain theraffinose family oligosaccharides. Almost all of the meals are utilizedas feed. However, human and animals, which do not have α-galactosidase,cannot directly digest the raffinose family oligosaccharides. It isknown that the raffinose family oligosaccharides lower the metabolicenergy efficiency of feed due to, for example, assimilation of theraffinose family oligosaccharides by enteric bacteria to cause gasproduction. It has been reported that removal of raffinose familyoligosaccharides from soybean meal results in a large increase in themetabolizable energy for broiler chickens (Coon, “Proceeding SoybeanUtilization Alternatives”, Univrsity of Minnesota, 203-211 (1989)). Inview of the foregoing facts, it is desired to develop the plants such assoybean, rapeseed, and cottonseed in which the raffinose familyoligosaccharides are decreased.

Such plants have been subjected to breeding to increase the amount ofoil. Photosynthetic products are distributed over oils, proteins, andcarbohydrates including the raffinose family oligosaccharides. It hasbeen reported for soybean that a reverse correlation exists between theamount of oils and the amount of carbohydrates. It is expected that thecontent of oils can be increased in a soybean plant having the samephotosynthetic ability as those possessed by others, by decreasing theproduction of the raffinose family oligosaccharides.

Based on a viewpoint as described above, Kerr et al. have reporteddevelopment of soybean varieties with a low content of the raffinosefamily oligosaccharides, by means of breeding based on mating andselection, in which the raffinose family oligosaccharides are lowered byan amount of 80% to 90% (WO 93/00742). However, this technique concernscreation of soybean variety, which cannot be applied to other varioussoybean varieties developed in response to, for example, aptitude forcultivation and resistance to disease. This technique cannot beuniversally applied to various plants as well.

It is known that raffinose, which is contained, for example, in sugarbeet and sugar cane, lowers crystal formation of sugar or sucrose.Therefore, it is possible to expect that if no raffinose is produced,the production efficiency of sugar may be improved in such a plant.However, no sugar beet has been created, which contains no raffinose.

As described above, the raffinose synthase, which has been hithertopurified, has been confirmed only as an enzyme activity, and no entityof the enzyme has been identified. The confirmed activity is low, and ithas been desired to obtain a raffinose synthase having a high activity.The conventional method for producing raffinose provides a low yield,and hence it has been desired to develop an efficient method forproducing raffinose. On the other hand, it is also desired to breed aplant in which the raffinose family oligosaccharides are decreased.

DISCLOSURE OF THE INVENTION

The present invention has been made taking the foregoing viewpoints intoconsideration, an object of which is to obtain a raffinose synthasehaving a high activity and DNA encoding raffinose synthase, and providean efficient method for enzymatically synthesis raffinose, and a methodfor utilizing DNA encoding raffinose synthase in plants.

As a result of diligent investigations in order to achieve the objectdescribed above, the present inventors have succeeded in purifying araffinose synthase from cucumber. Further diligent investigations havebeen made by the present inventors in order to clone a gene coding forthe raffinose synthase. As a result, a DNA fragment specific to a genefor the raffinose synthase has been obtained by chemically synthesizingsingle strand DNA's on the basis of nucleotide sequences deduced fromamino acid sequences of peptide fragments of the cucumber raffinosesynthase, and performing PCR by using the single strand synthetic DNA'sas primers and using cDNA's prepared from poly(A)⁺RNA extracted fromcucumber as templates. Further, the raffinose synthase gene has beenisolated by adopting a method in which hybridization is performed for acDNA library originating from cucumber by using the DNA fragment as aprobe. A chimeric gene having a regulatory region expressible in plantshas been prepared by using a fragment of the isolated raffinose synthasegene to transform a plant. Further, the function of endogenous raffinosesynthase has been regulated by introducing the raffinose synthase geneto create a plant in which the raffinose family oligosaccharides aredecreased.

Namely, the present invention provides a raffinose synthase which hasthe following properties:

-   -   (1) action and substrate specificity: the raffinose synthase        produces raffinose from sucrose and galactinol;    -   (2) optimum pH: the raffinose synthase has an optimum pH of        about 6 to 8;    -   (3) optimum temperature: the raffinose synthase has an optimum        temperature of about 35 to 40° C.;    -   (4) molecular weight: the raffinose synthase has:    -   (i) a molecular weight of about 75 kDa to 95 kDa estimated by        gel filtration chromatography;    -   (ii) a molecular weight of about 90 kDa to 100 kDa estimated by        polyacrylamide gel electrophoresis (Native PAGE); and    -   (iii) a molecular weight of about 90 kDa to 100 kDa estimated by        SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under a        reduced condition;    -   (5) inhibition: the raffinose synthase is inhibited by        iodoacetamide, N-ethylmaleimide, and myo-inositol.

In a specified embodiment of the foregoing raffinose synthase providedby the present invention, the raffinose synthase has an amino acidsequence including respective amino acid sequences shown in SEQ ID NOs.1 to 3 in Sequence Listing.

In another aspect of the present invention, there is provided araffinose synthase which is a protein specified by the following item(A) or (B):

-   -   (A) a protein which has an amino acid sequence shown in SEQ. ID        NO: 5 in Sequence Listing; or    -   (B) a protein which comprises an amino acid sequence including        substitution, deletion, insertion, addition, or inversion of one        or several residues of amino acids in the amino acid sequence        shown in SEQ ID NO: 5 in Sequence Listing, and which has an        activity to produce raffinose from sucrose and galactinol.

In still another aspect of the present invention, there is provided amethod for producing raffinose, comprising the step of allowing theforegoing raffinose synthase to act on sucrose and galactinol to produceraffinose.

In still another aspect of the present invention, there are provided DNAencoding raffinose synthase, and DNA coding for a protein specified bythe following item (A) or (B):

-   -   (A) a protein which has an amino acid sequence shown in SEQ ID        NO: 5 in Sequence Listing; or    -   (B) a protein which comprises an amino acid sequence including        substitution, deletion, insertion, addition, or inversion of one        or several residues of amino acids in the amino acid sequence        shown in SEQ ID NO: 5 in Sequence Listing, and which has an        activity to produce raffinose from sucrose and galactinol.

In a specified embodiment of the foregoing DNA of the present invention,there is provided DNA specified by the following item (a) or (b):

-   -   (a) DNA which includes a nucleotide sequence comprising at least        nucleotide residues having nucleotide numbers of 57 to 2408 in a        nucleotide sequence shown in SEQ ID NO: 4 in Sequence Listing;        or    -   (b) DNA which is hybridizable under a stringent condition with        the nucleotide sequence comprising at least nucleotide residues        having nucleotide numbers of 57 to 2408 in the nucleotide        sequence shown in SEQ ID NO: 4 in Sequence Listing, and which        codes for a protein having an activity to produce raffinose from        sucrose and galactinol.

In still another aspect of the present invention, there are provided achimeric gene comprising a raffinose synthase gene or a part thereof,and a transcription regulatory region expressible in plant cells, and aplant transformed with the chimeric gene.

In still another aspect of the present invention, there is provided amethod for changing a content of raffinose family oligosaccharides in aplant, comprising the steps of transforming the plant with the chimericgene, and expressing the gene in the plant.

In the following description, the raffinose synthase having theproperties described in the foregoing items (1) to (5), or the raffinosesynthase specified as the protein defined in the foregoing items (A) and(B) is simply referred to as “raffinose synthase” in some cases. DNAencoding raffinose synthase, or DNA encoding raffinose synthase andincluding non-translating regions is referred to as “raffinose synthasegene” in some cases.

The present invention will be explained in detail below.

<1> Raffinose Synthase of the Present Invention

The raffinose synthase of the present invention has the followingproperties:

-   -   (1) action and substrate specificity: the raffinose synthase        produces raffinose from sucrose and galactinol;    -   (2) optimum pH: the raffinose synthase has an optimum pH of        about 6 to 8;    -   (3) optimum temperature: the raffinose synthase has an optimum        temperature of about 35 to 40° C.;    -   (4) molecular weight: the raffinose synthase has:    -   (i) a molecular weight of about 75 kDa to 95 kDa estimated by        gel filtration chromatography;    -   (ii) a molecular weight of about 90 kDa to 100 kDa estimated by        polyacrylamide gel electrophoresis (Native PAGE); and    -   (iii) a molecular weight of about 90 kDa to 100 kDa estimated by        SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under a        reduced condition;    -   (5) inhibition: the raffinose synthase is inhibited by        iodoacetamide, N-ethylmaleimide, and myo-inositol.

The raffinose synthase having the foregoing properties has been isolatedand purified from leaves of cucumber, which has been identified for thefirst time by the present inventors. As demonstrated in Examplesdescribed later on, the raffinose synthase originating from cucumberincludes the respective amino acid residues shown in SEQ ID NOs: 1 to 3in Sequence Listing, in the amino acid sequence of the enzyme protein.An entire amino acid sequence of the raffinose synthase is shown in SEQID NO: 5.

The raffinose synthase is obtainable from plants belonging toCucurbitaceae, for example, plants such as melon (Cucumis melo) andcucumber (Cucumis sativus). Especially, the raffinose synthase iscontained in a large amount in leaves of these plants, especially intissues of leaf veins and seeds.

Next, the method for producing the raffinose synthase of the presentinvention will be explained in accordance with an illustrative methodfor isolating and purifying the raffinose synthase from cucumber.

Leaf vein tissues are collected from leaves of cucumber obtained 6 to 10weeks after planting. The vein are ground by a mortar with liquidnitrogen, to which a buffer is added to extract proteins. During thisprocess, it is allowable to add a substance to prevent the raffinosesynthase from degradation and inactivation, for example, a proteaseinhibitor such as PMSF (phenylmethanesulfonyl fluoride), or polyclarl AT(produced by Serva). Insoluble matters are removed from an obtainedextract solution by means of filtration and centrifugation to obtain acrude extract solution.

The crude extract solution thus obtained is subjected to fractionationbased on combination of ordinary methods for purifying proteins,including, for example, anion exchange chromatography, hydroxyapatitechromatography, gel filtration, and salting out. Thus the raffinosesynthase can be purified.

Anion exchange chromatography can be performed, for example, by using acolumn charged with a strongly basic anion exchanger such as HiTrap Q(produced by Pharmacia), or a weakly basic anion exchanger such asDEAE-TOYOPEARL (produced by Toyo Soda). The extract solution containingthe raffinose synthase is allowed to pass through the column so that theenzyme is adsorbed to the column. After washing the column, the enzymeis eluted by using a buffer having a high salt concentration. Duringthis process, the salt concentration may be increased in a stepwisemanner, or the concentration gradient may be applied. For example, whenthe HiTrap Q column is used, the raffinose synthase activity adsorbed tothe column is eluted by NaCl at about 0.3 M. An eluting solution to givean NaCl concentration gradient of 0.05 M to 0.35 M is preferably usedfor DEAE-TOYOPEARL. An eluting solution to give a phosphateconcentration gradient of 0.01 M to 0.3 M is preferably used forhydroxyapatite chromatography.

The order of the foregoing operations is not specifically limited. Eachof the operations may be repeated two or more times. It is desirable toexchange a sample solution with an appropriate buffer by means ofdialysis or the like before the sample solution is allowed to passthrough each column. The sample solution may be concentrated at eachstage.

At each stage of the purification, it is preferable that the raffinosesynthase activity contained in each of fractionated fractions ismeasured so that fractions having high activities are collected to beused in the next stage. The method for measuring the raffinose synthaseactivity is exemplified by a method based on the use of radioisotope asreported, for example, by Lehle, H. et al. (Eur. J. Biochem., 38,103-110 (1973)). As a modified method thereof, the reaction temperatureand the substrate concentration may be changed. For example, 10 μl of anenzyme solution is added to a reaction solution containing, at finalconcentrations, 10 mM ¹⁴C-sucrose, 20 mM galactinol, 25 mM HEPES(2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid)-NaOH, pH 7.0,0.5 mM DTT (dithiothreitol) to give a volume of 50 μl. The solution isincubated at 32° C. for 1 hour to perform the reaction. The reaction isstopped by adding 200 μl of ethanol and heating the solution at 95° C.for 30 seconds. The reaction solution is centrifuged to obtain asupernatant. An aliquot of the supernatant is spotted on Whatman 3MMfilter paper, and developed with n-propanol: ethyl acetate: water=4:1:2.Incorporation of ¹⁴C into raffinose is investigated, which is regardedto be the raffinose synthase activity (nmol/hour).

The present inventors have developed a method for measuring theraffinose synthase activity in place of the foregoing method. Namely,the raffinose synthase activity is measured by quantitativelydetermining raffinose produced by the raffinose synthesis reaction, bymeans of HPLC (high-performance liquid chromatography). According tothis method, the activity can be measured conveniently and quickly ascompared with the method of Lehle, H. et al. This method is especiallypreferable to detect active fractions during the purification operation.This method will be explained below.

The raffinose synthesis reaction is based on the use of a reactionsolution prepared to have a composition having the following finalconcentrations. The reaction solution is added with 10 to 50 μl of araffinose synthase solution to give a volume of 100 μl, followed byperforming the reaction at 32° C. for 60 minutes.

[Composition of Reaction Solution (Final Concentration)]

-   -   2.5 mM sucrose    -   5 mM galactinol    -   5 mM DTT    -   20 mM Tris-HCl buffer (pH 7.0)

After performing the reaction as described above, the reaction solutionis added with ethanol in a volume four times the volume of the reactionsolution to stop the reaction by heating the solution at 95° C. for 30seconds. The obtained solution is centrifuged to obtain a supernatantwhich is then dried up under a reduced pressure. After that, an obtainedresidue is dissolved in distilled water. Raffinose in the reactionproduct is quantitatively determined by using HPLC to estimate theraffinose synthase activity. HPLC can be performed by using, forexample, Sugar Analysis System DX500 (CarboPac PA1 column, pulsedamperometry detector (produced by Dionecs)).

FIG. 1 shows a result of measurement performed in accordance with themethod described above, for the amount of raffinose produced when thereaction time was changed. As clarified from FIG. 1, this method makesit possible to conveniently measure the raffinose synthase activity withexcellent linearity.

The degree of purification of the purified raffinose synthase can beconfirmed, and the molecular weight can be measured, by means of, forexample, gel electrophoresis and gel filtration chromatography.Enzymatic properties can be investigated by measuring the enzymeactivity while changing the reaction temperature or the reaction pH, orby measuring the remaining enzyme activity after adding, to the reactionsolution, various enzyme inhibitors, metal ions or the like. The stablepH range and the stable temperature range can be investigated bymeasuring the enzyme activity after exposing the raffinose synthase tovarious pH conditions and temperature conditions for a certain period oftime respectively.

The properties of the raffinose synthase described above have beendetermined in accordance with procedures as described above. However, itshould be noted that different results may be obtained depending onmeasurement conditions. For example, the measurement for the molecularweight based on the use of gel filtration chromatography is affected bythe type of the gel filtration agent and the buffer, and the molecularweight marker to be used. The enzyme activity differs depending on thetype of the buffer and the salt concentration in many cases even whenthe measurement is performed at an identical pH. Therefore, uponidentification for the raffinose synthase, it is preferable to performcomprehensive investigation without being bound to only measurement forindividual properties.

The raffinose synthase of the present invention is obtained byperforming the isolation and purification from cucumber as describedabove. Alternatively, the raffinose synthase of the present inventioncan be produced by introducing, into an appropriate host, DNA coding forthe raffinose synthase described later on, and making expressionthereof, in accordance with ordinary methods used for fermentativeproduction of heterogeneous proteins.

Those assumed as the host for expressing the raffinose synthase geneinclude various procaryotic cells represented by Escherichia coli, andvarious eucaryotic cells represented by Saccharomyces cerevisiae.However, it is desirable to use plant cells, especially cellsoriginating from plants such as tobacco, cucumber, and Arabidopsisthaliana.

The recombinant plasmid used for transformation can be prepared byinserting DNA coding for the raffinose synthase into an expressionvector in conformity with the type of cells to be used for expressiontherein. Those usable as the plant expression vector include thosehaving a promoter DNA sequence capable of being expressed in the plantor a combination of a plurality of such promoter DNA sequences, and aterminator DNA sequence workable in the plant, and further having asequence between the both to make it possible to insert a foreign gene.

The promoter includes, for example, promoters which make expression overa whole plant, such as CaMV 35S RNA promoter, CaMV 19S RNA promoter, andnopaline synthase promoter; promoters which make expression in greentissues, such as Rubisco small subunit promoter; and promoters whichmake site-specific expression at portions such as seed, including, forexample, those for genes of napin and phaseolin. The terminatordescribed above includes, for example, nopaline synthase terminator, andRubisco small subunit 3′-side portion.

As for the expression vector for plants, for example, pBI121 andp35S-GFP (produced by CLONTECH) are commercially available, which may beused. Alternatively, a vector for expressing virus RNA may be used sothat a gene for an outer coat protein encoded thereby, for example, maybe replaced with the raffinose synthase gene.

In order to achieve transformation, it is advantageous to use methodswhich are usually used for transformation, such as the Agrobacteriummethod; the particle gun method, the electroporation method, and the PEGmethod, in conformity with a host cell to be manipulated. The raffinosesynthase activity can be detected by using the method adopted in thepurification process for the raffinose synthase. Upon the detection, itis desirable to previously remove α-galactosidase, for example, byallowing the sample to pass through an anion exchange column.

The gene coding for the raffinose synthase originating from cucumberincludes all of those which provide the raffinose synthase activity uponexpression. Preferably, the gene is exemplified by the gene comprisingDNA coding for the amino acid sequence shown in SEQ ID NO: 5 in SequenceListing, and the gene having the nucleotide sequence shown in SEQ ID NO:4 in Sequence Listing. It is noted that the gene coding for the aminoacid sequence shown in SEQ ID NO: 5 in Sequence Listing includes variousnucleotide sequences taking degeneracy of codons into consideration.Namely, the gene coding for the amino acid sequence shown in SEQ ID NO:5 in Sequence Listing may be selected from such various nucleotidesequences, while considering several factors for the gene expressionsystem, such as preferential codons depending on, for example, the typeof the host cell, and avoidance of higher-order structure to be formedby transcribed RNA. The selected nucleotide sequence may be DNA clonedfrom the nature, or DNA chemically synthesized in an artificial manner.

<2> DNA Coding for Raffinose Synthase of the Present Invention

DNA coding for the raffinose synthase can be obtained by preparing acDNA library from poly(A)⁺RNA isolated from a plant such as cucumber,and screening the cDNA library by means of hybridization. A probe to beused for the hybridization can be obtained by performing amplificationby means of PCR (polymerase chain reaction) by using, as primers,oligonucleotides synthesized on the basis of partial amino acidsequences of the raffinose synthase protein.

A method for obtaining DNA of the present invention from poly(A)⁺RNAoriginating from cucumber will be specifically explained below.

As for the position for extractiing poly(A)⁺RNA, all portions of acucumber plant body may be used provided that the raffinose synthasegene is expressed at that portion. poly(A)⁺RNA can be obtained, forexample, from leaves, stalks, buds, fruits, and seeds at various growthstages. However, poly(A)⁺RNA is desirably obtained from a material offully expanded leaves after fruiting, especially leaf vein portions.

In order to extract total RNA from the cucumber tissue, any method maybe used without limitation provided that RNA can be efficiently obtainedwith less damage. It is possible to use any known method such as thephenol/SDS method and the guanidine isothiocyanate/cesium chloridemethod. Poly(A)⁺RNA can be isolated from the total RNA thus obtained, byusing an oligo(dT) cellulose. It is also preferable to use a kit (forexample, MPG Direct mRNA Purification Kit, produced by CPG, INC.) whichmakes it possible to obtain poly(A)⁺RNA without extracting the totalRNA.

A DNA fragment, which is used as a probe for screening for the cDNAlibrary, can be obtained by performing PCR. Oligonucleotides, which havenucleotide sequences deduced from already known amino acid sequences ofpeptide fragments, for example, nucleotide sequences deduced from aminoacid sequences shown in SEQ ID NOs: 1 to 3, are chemically synthesized.The obtained oligonucleotides are used as primers to perform PCR. Anyportion of the amino acid sequence of the obtained peptide fragment maybe used for the primers. However, it is desirable to select sequenceswhich include less degeneracy of codons and which are assumed to form nocomplicated higher-order structure. Alternatively, it is also preferableto perform RACE (Rapid Amplification of cDNA End, “PCR PROTOCOLS A Guideto Methods and Applications”, ACADEMIC press INC., pp. 28 to 38).

It is desirable to use, as a template for PCR, a cDNA library or singlestrand cDNA. When heat-resistant DNA polymerase having a reversetranscriptase activity is used for the PCR reaction, it is allowable touse poly(A)⁺RNA, or total RNA in some cases.

In order to prepare the cDNA library, at first single strand cDNA's aresynthesized by using reverse transcriptase while using poly(A)⁺RNA as atemplate and using oligo(dT) primer and random primers. Next, doublestrand cDNA's are synthesized in accordance with, for example, theGubler and Hoffman method, the Okayama-Berg method (“Molecular Cloning”,2nd edition, Cold Spring Harbor press, 1989). When the raffinosesynthase gene is expressed in a small amount, cDNA's may be amplified bymeans of PCR by using a cDNA library construction kit based on the useof PCR (for example, Capfinder PCR cDNA Library Construction Kit(produced by CLONTECH)). cDNA's thus synthesized can be cloned into acloning vector such as phage vectors and plasmids, after performing, forexample, blunt end formation, addition of linker, addition ofrestriction enzyme site by means of PCR.

A portion characteristic of the raffinose synthase cDNA is selected fromthe DNA fragments obtained by PCR described above, for the probe forhybridization. It is desirable to select a DNA fragment located near tothe 5′-terminal side. The amplified DNA fragment thus selected ispurified from a reaction solution of PCR. In this procedure, theamplified DNA fragment may be purified by subcloning the DNA fragment byusing a plasmid, preparing a large amount of a subcloned plasmid whichis thereafter digested with a restriction enzyme, and excising the DNAfragment from a gel after electrophoresis. Alternatively, the amplifiedDNA fragment may be purified by performing PCR by using the plasmid as atemplate to amplify and use only the objective portion. When the amountof the initially amplified DNA fragment is sufficiently large, theamplified DNA fragment may be purified by electrophoresing the DNAfragment without performing subcloning, excising a gel segmentcontaining a band of the objective DNA fragment, and purifying the DNAfragment from the gel segment.

Screening to obtain the objective clone from the cDNA library isperformed by means of hybridization. The DNA fragment obtained inaccordance with the foregoing method can be labeled and used as a probefor the hybridization. Upon labeling, it is possible to use variouslabels such as radioisotope and biotin. However, labeling is desirablyperformed in accordance with the random priming method. Screening may beperformed by using PCR instead of hybridization. Further, screening maybe performed by using hybridization and PCR in combination.

The nucleotide sequence of DNA coding for the raffinose synthaseoriginating from cucumber obtained as described above, and the aminoacid sequence deduced from the nucleotide sequence are illustrativelyshown in SEQ ID NO: 4 in Sequence Listing. Only the amino acid sequenceis shown in SEQ ID NO: 5. A transformant AJ13263 of Escherichia coliJM109, which harbors a plasmid pMossloxCRS containing the DNA fragmentincluding DNA coding for the raffinose synthase obtained in Example 3described later on, has been internationally deposited on the basis ofthe Budapest Treaty since Nov. 19, 1996 in National Institute ofBioscience and Human Technology of Agency of Industrial Science andTechnology of Ministry of International Trade and Industry (postal code:305, 1-3 Higashi-Icchome, Tsukuba-shi, Ibaraki-ken, Japan), and awardeda deposition number of FERM BP-5748.

The DNA of the present invention may code for a raffinose synthaseprotein including substitution, deletion, insertion, addition, orinversion of one or several residues of amino acids at one or severalpositions, provided that the activity of raffinose synthase encodedthereby, i.e., the activity to produce raffinose from sucrose andgalactinol is not deteriorated. In this context, the number of “severalresidues” differs depending on the position and the type of the aminoacid residues in the three-dimensional structure of the protein,originally because of the following reason. Namely, high similarity isfound between some amino acids and other amino acids, for example,between isoleucine and valine, and such a difference in amino acid doesnot greatly affect the three-dimensional structure of the protein.Therefore, the DNA of the present invention may code for those havinghomology of not less than 35 to 40% with respect to the entire 784 aminoacid residues for constructing the raffinose synthase originating fromcucumber, provided that they have the raffinose synthase activity.Preferably, those encoded by the DNA of the present invention havehomology of 65% in a region between 510th amino acid and 610th aminoacid. Specifically, the number of “several residues” is 2 to 40,preferably 2 to 20, and more preferably 2 to 10.

The present invention includes genes in which homology of not less thanabout 50% is given for the entire length of the gene, and homology ofnot less than 65% is given over a region comprising about 300 nucleotideresidues. Nucleotide sequence information on such genes can be obtainedby searching genes having homology to the raffinose synthase geneoriginating from cucumber, by using data base such as GenBank. Forexample, GENETIX-MAC (software for processing genetic information,produced by Software Development), which adopts the Lipman-Personmethod, may be used as a program for homology analysis. Alternatively,those open to the public on the Internet may be used for this purpose.Some nucleotides sequences obtained by the method as described abovecontain the entire length of the gene, and other nucleotide sequences donot contain the entire length of the gene. When the entire length of thegene is not contained, the entire length gene can be easily obtained byusing RNA extracted from an objective plant tissue as a template, andusing primers corresponding to portions having high homology to theraffinose synthase gene originating from cucumber, in accordance withthe 5′-RACE method and the 3′-RACE method. The obtained entire lengthgene may be incorporated into an appropriate expression vector providedas those included in a kit such as Soluble Protein Expression System(produced by INVITROGEN), Tight Control Expression System (produced byINVITROGEN), and QIAexpress System (produced by QIAGEN) so that the genemay be expressed. The raffinose synthase activity may be measured inaccordance with the method described above to select a clone having theactivity.

DNA, which codes for substantially the same protein as the raffinosesynthase, is obtained by modifying the nucleotide sequence in accordancewith, for example, the site-directed mutagenesis method so that aminoacids located at specified positions are subjected to substitution,deletion, insertion, or addition.

Modified DAN as described above may be also obtained in accordance withthe conventionally known mutation treatment. The mutation treatmentincludes a method in which the DNA coding for the raffinose synthase istreated with hydroxylamine or the like in vitro, and a method in which abacterium belonging to the genus Escherichia harboring the DNA codingfor the raffinose synthase gene is treated with ultraviolet irradiationor a mutating agent usually used for artificial mutation, such asnitrous acid and N-methyl-N′-nitro-N-nitrosoguanidine (NTG).

The substitution, deletion, insertion, addition, or inversion of thenucleotide includes mutation which naturally occurs, for example, basedon the difference between individuals of a cucumber plant, thedifference between varieties, the formation of multiple copies of thegene, the difference between respective organs, and the differencebetween respective tissues.

DNA having mutation as described above is expressed in an appropriatecell to investigate the raffinose synthase activity of an expressedproduct. Thus it is possible to obtain DNA which codes for substantiallythe same protein as the raffinose synthase. Further, DNA coding forsubstantially the same protein as the raffinose synthase protein can beobtained by isolating DNA which is hybridizable under a stringentcondition with DNA having a nucleotide sequence comprising nucleotideresidues having, for example, nucleotide numbers of 56 to 2407 in thenucleotide sequence shown in SEQ ID NO: 4 in Sequence Listing, and whichcodes for the protein having the raffinose synthase activity, from DNA'scoding for raffinose synthases having mutation or from cells harboringthe DNA's. The phrase “stringent condition” referred to herein indicatesa condition in which so-called specific hybrid is formed, andnonspecific hybrid is not formed. It is difficult to definitely expressthis condition by using numerical values. However, for example, thiscondition includes a condition in which DNA's having high homology, forexample, DNA's having homology of not less than 50% hybridize with eachother, while DNA's having homology lower than the above do not hybridizewith each other, or a condition in which hybridization is achieved at asalt concentration corresponding to a washing condition for ordinarySouthern hybridization, i.e., 1×SSC, 0.1% SDS, and preferably 0.1×SSC,0.1% SDS at 60° C. Genes, which hybridize under such a condition,include those which contain a stop codon generated at an intermediateposition, and those which have lost the activity due to mutation at theactive center. However, those having such inconvenient mutation can beeasily eliminated by ligating the gene with a commercially availableactivity expression vector to measure the raffinose synthase activity inaccordance with the method described above.

When the DNA of the present invention is used to express antisense RNAfor the raffinose synthase, it is unnecessary for the DNA to code forany active raffinose synthase. Further, the function of any endogenousgene having homology can be restrained by using sense RNA. In such acase, it is also unnecessary for the DNA to code for any activeraffinose synthase. Further, it is unnecessary for the DNA to containthe entire length. Preferably, it is sufficient for the DNA to haveabout 500 base pairs of an N-terminal side translating region having 60%of homology.

The method has been explained above, in accordance with which thepresent inventors have succeeded in cloning the objective cDNA of theraffinose synthase originating from cucumber. However, other than theforegoing, the following methods may be available.

(1) The raffinose synthase originating from cucumber is isolated andpurified, and an entire nucleotide sequence is chemically synthesized onthe basis of a determined amino acid sequence or the amino acid sequenceshown in SEQ ID NO: 5.

(2) Chromosomal DNA is prepared from a cucumber plant body, and achromosomal DNA library is prepared by using a plasmid vector or thelike. The raffinose synthase gene is obtained from the library by meansof hybridization or PCR. It is assumed that the raffinose synthase geneoriginating from chromosome contains intron in its coding region.However, DNA divided into several parts by such intron is included inthe DNA of the present invention provided that it codes for theraffinose synthase.

(3) Poly(A)⁺RNA is fractionated into fractions in accordance with themolecular weight or the like. The fractions are subjected to an in vitrotranslation system based on the use of wheat germ or rabbit reticulocyteto determine a fraction containing-mRNA coding for a polypeptide havingthe raffinose synthase activity. An objective cDNA fragment is preparedand obtained from the fraction.

(4) An anti-cucumber raffinose synthase antibody is prepared. Elementsof a cDNA library are incorporated into a protein expression vector, andan appropriate host is infected therewith to express proteins encoded bycDNA's. An objective cDNA may be screened by using the foregoingantibody.

(5) Appropriate primers are synthesized on the basis of amino acidsequences of peptide fragments, and a sequence containing the terminalis amplified by means of the RACE method, followed by cloning thereof.

<3> Method for Producing Raffinose of the Present Invention

In the method for producing raffinose of the present invention, theraffinose synthase is allowed to act on sucrose and galactinol toproduce raffinose. When the raffinose synthase is allowed to act onsucrose and galactinol, the galactose residue used for constructinggalactinol is transferred to sucrose, and thus raffinose is produced.During this process, myo-inositol used for constructing galactinol isliberated.

The raffinose synthase, which is used to produce raffinose, may be anenzyme extracted from a plant body, or an enzyme produced by means ofthe genetic recombination technique based on the use of the DNA of thepresent invention.

In order to allow the raffinose synthase to act on sucrose andgalactinol, the following procedure may-be available. Namely, theraffinose synthase or cells having an ability to produce the raffinosesynthase are immobilized to a carrier such as alginic acid gel andpolyacrylamide gel to prepare immobilized enzyme or immobilized cells.The immobilized enzyme or the immobilized cells are charged to a column,and a solution containing sucrose and galactinol is allowed to passthrough the column. As for the carrier and the method for immobilizingthe raffinose synthase or the cells to the carrier, it is possible toadopt materials and methods which are used for ordinary bioreactors.

The raffinose synthesis reaction is performed, for example, by addingthe raffinose synthase to a solution such as an aqueous solution or abuffer containing sucrose and galactinol. It is preferable that pH ofthe solution is adjusted to be within a range of about 6 to 8,especially at about pH 7. The reaction temperature is within a range ofabout 28 to 42° C., preferably 35 to 40° C., especially about 38° C. Theraffinose synthase of the present invention is stable within a range ofpH 5 to 8, especially in the vicinity of pH 6. The enzyme of the presentinvention is stable within a temperature range of not more than about40° C.

The enzyme activity of the raffinose synthase of the present inventionis inhibited by iodoacetamide, N-ethylmaleimide, MnCl₂, ZnCl₂, andNiCl₂. Therefore, it is desirable that these substances are notcontained in the reaction solution.

Preferably, galactinol and sucrose are added to the reaction solution ata concentration of not less than 5 mM of galactinol and a concentrationof not less than 1.5 mM of sucrose. The raffinose synthase may be addedto the reaction solution in an amount depending on the amounts of thesubstrates.

Raffinose is separated from unreacted sucrose and galactinol and frommyo-inositol produced by the enzyme reaction, contained in the reactionsolution, in accordance with a method including, for example, gelfiltration chromatography.

<4> Chimeric Gene and Transgenic Plant of the Present Invention

The chimeric gene of the present invention includes the raffinosesynthase gene or a part thereof and the transcription regulatory regionexpressible in plant cells. The raffinose synthase gene is exemplifiedby the DNA coding for the raffinose synthase of the present inventiondescribed in the foregoing item <2>. When the chimeric gene of thepresent invention is used as an antisense gene, a non-coding region ofthe raffinose synthase gene or a part thereof can be used in some cases,besides the DNA coding for the raffinose synthase. The non-coding regionincludes, for example, sequences indicated by nucleotide numbers of 1 to55 (5′-non-coding region) and nucleotide numbers of 2407 to 2517(3′-non-coding region) in SEQ ID NO: 4 in Sequence Listing.

When the transcription regulatory region is linked to the DNA coding forthe raffinose synthase in the chimeric gene of the present invention sothat mRNA (sense RNA) homologous to the coding strand of the DNA isexpressed, plant cells introduced with the chimeric gene express theraffinose synthase, and the content of the raffinose familyoligosaccharides is increased. On the other hand, when thetranscriptional regulatory region is linked to the DNA so that RNA(antisense RNA) having a sequence complementary to the coding strand ofthe DNA is expressed, and when the transcription regulatory region islinked to the DNA so that a partial fragment of the raffinose synthasegene, preferably sense RNA for a portion of not less than about 200 basepairs in the upstream coding region is expressed, then the expression ofendogenous raffinose synthase is restrained in plant cells introducedwith the chimeric gene, and the raffinose family oligosaccharides aredecreased.

The content of the raffinose family oligosaccharides in a plant can bechanged by transforming the plant with the chimeric gene of the presentinvention, and expressing the gene in cells of the plant.

Plants to which the present invention is applicable include, forexample, oil crops such as soybean, rapeseed, cotton; sugar crops suchas sugar beet and sugar cane; and model plants represented byArabidonsis thaliana.

The transcription regulatory region expressible in plant cells includes,for example, promoters which make expression over a whole plant, such asCaMV 35S RNA promoter, CaMV 19S RNA promoter, and nopaline synthasepromoter; promoters which make expression in green tissues, such asRubisco small subunit promoter; and promoter regions which makesite-specific expression at portions such as seed, including, forexample, those for genes of napin and phaseolin as described above. The3′-terminal of the chimeric gene may be connected with the terminatorsuch as nopaline synthase terminator, and Rubisco small subunit 3′-endportion.

The plant may be transformed with the chimeric gene in accordance withusually used methods such as the Agrobacterium method, the particle gunmethod, the electroporation method, and the PEG method, depending on thehost cell to be manipulated.

The transformation method for introducing the chimeric gene into theplant includes, for example, the Agrobacterium method, the particle gunmethod, the electroporation method, and the PEG method.

The Agrobacterium method is specifically exemplified by a method basedon the use of a binary vector. Namely, a plant is infected with a vectorcomprising T-DNA originating from Ti plasmid, a replication origin whichis functional in microorganisms such as Escherichia coli, and a markergene for selecting plant cells or microbial cells harboring the vector.Seeds are collected from the plant, and they are allowed to grow. Plantsintroduced with the vector are selected by using an index of expressionof the marker gene. Obtained plants are measured for the raffinosesynthase activity, or strains exhibiting change in content of theraffinose family oligosaccharides are selected from the obtained plants.Thus it is possible to obtain an objective transformed plant.

A method for introducing the chimeric gene into soybean will bedescribed below. In order to perform transformation for soybean, it ispossible to use any one of the particle gun method (Pro. Natl. Acad.Sci. USA, 86, 145 (1989); TIBTECH, 8, 145 (1990); Bio/Technology, 6, 923(1988); Plant Physiol, 87, 671 (1988); Develop. Genetics, 11, 289(1990); and Plant cell Tissue & Organ Culture, 33, 227 (1993)), theAgrobacterium method (Plant Physiol., 91, 1212 (1989); WO 94/02620;Plant Mol. Biol., 9, 135 (1987); and Bio/Technology, 6, 915 (1933)), andthe electroporation method (Plant Physiol, 99, 81 (1992); Plant Physiol,84, 856 (1989); and Plant Cell Reports, 10, 97 (1991)).

In the particle gun method, it is preferable to use an embyogenic tissueor a hypocotyl of an immature seed about 30 to 40 days after dehiscenceof anthesis. About 1 g of the embyogenic tissue is spread over a petridish, into which, for example, gold particles or tungsten particlescoated with the objective chimeric gene may be shot. The tissue istransferred after 1 to 2 hours to a liquid medium to performcultivation. After 2 weeks, the tissue is transferred to a mediumcontaining an antibiotic for transformant selection, followed bycultivation. After 6 weeks, a green adventitious embryo which isresistant to the antibiotic is obtained. The adventive embryo is furthertransferred to a fresh medium and cultured so that a plant body isreproduced. Alternatively, when the hypocotyl is used, the hypocotyl isexcised under a sterilized condition, and it is treated in accordancewith the particle gun method, followed by cultivation in MS medium(Murashige and Skoog, Physiologia Plantrum, 15, 473-497 (1962))containing cytokinin at a high concentration. The hypocotyl is culturedin the darkness for 2 weeks, and then it is cultured at room temperaturewith light irradiation for 12 to 16 hours in MS medium having a loweredcytokinin content. During this process, it is preferable to add, to themedium, the antibiotic having been used as the selection marker. When amultiple bud body is formed from the transplanted tissue, it istransferred to a medium added with no hormone so that rooting is caused.An obtained seedling body is transferred to a greenhouse and cultivated.

In the case of the method based on the use of Agrobacterium, it isdesirable to use Cotyldonary nod as a plant tissue. Commerciallyavailable LBA4404, C58, and Z707 can be used as Agrobacterium. However,it is desirable to use Z707. For example, a plasmid obtained byinserting the objective gene into pMON530 (produced by Monsanto Co.) canbe used as the vector. The plasmid is introduced into Agrobacteriumtumefaciens Z707 (Hepburn et al., J. Gen. Microbiol., 131, 2961 (1985))in accordance with, for example the Direct freeze thaw method (An etal., “Plant Mol. Biol. Mannual”, A3: 1-19, 1988). The Agrobacteriumtransformed with the chimeric gene is cultivated overnight. Proliferatedcells are collected by centrifugation at 5000 rpm for 5 minutes, andthey are suspended in B5 suspension medium. Soybean seeds aresterilized, and they are cultivated for 3 days on B5 medium having a{fraction (1/10)} concentration so that they germinate. Cotyledons areexcised, and they are cultivated for 2 hours with the suspension ofAgrobacterium. The cotyledons are transferred to B5 medium (containingGamborg B5 salt (Exp. Cell. Res., 50, 151 (1968)), Gamborg B5 vitamin,3% sucrose, 5 μM benzylaminopurine, 10 μM IBA, and 100 μMacetosyringon), and they are cultivated for 3 days under a condition at25° C. with light irradiation (60 μEm⁻²S⁻¹) for 23 hours. Subsequently,in order to remove Agrobacterium, the cotyledons are cultivated in B5medium (5 μM benzylaminopurine, 10 mg/L carbenicillin, 100 mg/Lvancomycin, and 500 mg/L cefotaxime) at 25° C. for 4 days whileexchanging the medium every day. After that, the cotyledons arecultivated in B5 medium (200 mg/L kanamycin). Multishoots are formedwithin 1 or 2 months. They are cultivated on B5 medium (0.58 mg/Lgibberellin and 50 mg/L kanamycin) to elongate the shoots. Subsequently,the shoots are transferred to B5 medium (10 μM IBA) to cause rooting.Rooted seedlings are acclimatized, and they are cultivated in agreenhouse. Thus transformants can be obtained.

A transformant plant, in which the raffinose synthase gene isintroduced, can be easily confirmed by extracting DNA from thetransformant, and performing Southern hybridization by using theraffinose synthase gene as a probe.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a relationship between the reaction time and the amount ofraffinose produced by the raffinose synthesis reaction.

FIG. 2 shows a photograph illustrating a result of SDS-polyacrylamidegel electrophoresis for the raffinose synthase. M indicates molecularweight markers, and S indicates a sample containing the raffinosesynthase. Numerals indicate molecular weights (kDa).

FIG. 3 shows an influence of the reaction temperature on the raffinosesynthase activity.

FIG. 4 shows an influence of the reaction pH on the raffinose synthaseactivity.

FIG. 5 shows an influence of myo-inositol on the raffinose synthaseactivity.

FIG. 6 shows a stable pH range of the raffinose synthase.

FIG. 7 shows relationships between synthetic primers and amino acidsequences of peptides. R represents A or G, Y represents C or T, Mrepresents A or C, K represents G or T, D represents G, A, or T, Hrepresents A, T, or C, B represents G, T, or C, N represents G, A, T, orC, and I represents inosine.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be more specifically explained below withreference to Examples.

At first, the method for measuring the raffinose synthase activity, usedto confirm active fractions during respective purification steps andinvestigate characteristics of the enzyme in the following Examples,will be explained.

<Method for Measuring the Raffinose Synthase Activity>

The activity of the raffinose synthase was measured by quantitativelydetermining raffinose produced by the raffinose synthesis reaction byusing HPLC (high-performance liquid chromatography). HPLC was performedby using Sugar Analysis System DX500 (CarboPac PA1 column, pulsedamperometry detector (produced by DIONEX)).

The raffinose synthesis reaction was performed by using a reactionsolution prepared to have a composition having the following finalconcentrations. The reaction solution was added with 10 to 50 μl of araffinose synthase solution to give a volume of 100 μl, followed byperforming the reaction at 32° C. for 60 minutes.

[Composition of Reaction Solution (Final Concentration)]

-   -   2.5 mM sucrose    -   5 mM galactinol    -   5 mM DTT    -   20 mM Tris-HCl buffer (pH 7.0)

After performing the reaction as described above, the reaction solutionwas added with ethanol in a volume four times the volume of the reactionsolution to stop the reaction by heating the solution at 95° C. for 30seconds. The obtained solution was centrifuged to obtain a supernatantwhich was then dried up under a reduced pressure. After that, anobtained residue was dissolved in distilled water. Raffinose in thereaction product was quantitatively determined by using the sugaranalysis system to estimate the raffinose synthase activity.

EXAMPLE 1 Purification of Raffinose Synthase from Cucumber

<1> Extraction of Raffinose Synthase from Cucumber

Vein tissues were collected from true leaves of cucumber (cv.: SUYOU)obtained 6 to 10 weeks after planting. The leaf vein tissues were frozenwith liquid nitrogen, and they were stored at −80° C. The frozen leafvein tissues were ground by a mortar with liquid nitrogen, to whichBuffer 1 (40 mM Tris-HCl buffer (pH 7.0), 5 mM DTT, 1 mM PMSF(phenylmethanesulfonyl fluoride), 1% polyclarl AT (produced by Serva))was added to extract proteins. An obtained extract solution wasfiltrated with a filter such as gauze or Miracloth (produced byCalbiochem-Novobiochem). An obtained filtrate was centrifuged at 4° C.at about 30,000×g for 60 minutes. A supernatant obtained by thecentrifugation was used as a crude extract solution.

<2> Anion Exchange Chromatography (1)

The crude extract solution (about 560 ml) obtained as described abovewas applied to a column system comprising five connected columns forstrongly basic anion exchange chromatography (HiTrap Q, produced byPharmacia, 1.6 cm×2.5 cm) equilibrated with Buffer 2 (20 mM Tris-HClbuffer (pH 7.0), 5 mM DTT) to adsorb the raffinose synthase activity tothe columns. Subsequently, the columns were washed with Buffer 3 (20 mMTris-HCl buffer (pH 7.0), 0.2 M NaCl, 5 mM DTT) in a volume five timesof the columns so that non-adsorbed proteins were washed out. Afterthat, the raffinose synthase activity was eluted from the columns with50 ml of Buffer 4 (20 mM Tris-HCl buffer (pH 7.0), 0.3 M NaCl, 5 mMDTT).

<3> Anion Exchange Chromatography (2)

The eluted solution (about 75 ml) was placed in a dialysis tube(Pormembranes MWC 0:10,000, produced by Spectra), and it was dialyzedagainst 10 L of Buffer 5 (20 mM Tris-HCl buffer (pH 7.0), 0.05 M NaCl, 5mM DTT) at 4° C. overnight. The dialyzed sample was applied to a columnfor weakly basic anion exchange chromatography (DEAE-TOYOPEARL, producedby Toyo Soda, 2.2×20 cm) equilibrated with Buffer 5 to adsorb theraffinose synthase activity to the column. Subsequently, the column waswashed with Buffer 5 in a volume five times the volume of the column towash out non-adsorbed proteins. After that, a linear concentrationgradient of 0.05 M to 0.35 M NaCl in a volume twenty times the volume ofthe column was applied to elute the enzyme activity so thatfractionation was performed.

<4> Gel Filtration Chromatography

The eluted solution obtained as described above (about 160 ml) wasconcentrated into 6.5 ml by using a concentrator (Centriprep 10,produced by Amicon). Aliquots (each 3 ml) of the concentrated solutionwere applied to a column for gel filtration chromatography (Superdex 200pg, produced by Pharmacia, 2.6 cm×60 cm). Equilibration for the columnand elution from the column were performed by using Buffer 6 (20 mMTris-HCl buffer (pH 7.0), 0.1 M NaCl, 5 mM DTT, 0.02% Tween 20).Fractions having the raffinose synthase activity were collected fromfractionated fractions.

<5> Hydroxyapatite Chromatography

A collected fraction (about 25 ml) having the raffinose synthaseactivity fractionated by the gel filtration was concentrated by usingCentriprep 10, and the buffer was exchanged with Buffer 7 (0.01 M sodiumphosphate buffer (pH 7.0), 5 mM DTT, 0.02% Tween 20). An obtainedconcentrate solution (about 1.2 ml) was applied to a hydroxyapatitecolumn (Bio-Scale CHT-1, produced by Bio Rad, 0.7×5.2) previouslyequilibrated with the same buffer to adsorb the raffinose synthaseactivity. The column was washed with the same buffer in a volume (10 ml)five times the volume of the column. After that, a linear concentrationgradient of 0.01 M to 0.3 M phosphate in a volume twenty times thevolume of the column was applied to elute the enzyme activity so thatfractionation was performed.

<6> Hydroxyapatite Rechromatography

An active fraction obtained in accordance with the hydroxyapatitechromatography as described above was subjected to rechromatography inthe same manner as described above to obtain a purified raffinosesynthase fraction (about 2 ml).

The amount of protein contained in the active fraction was about 200 μg.The total activity was 5700 nmol/hour, and the specific activity perprotein was 28 μmol/hour/mg. The active fraction contained only aprotein which exhibited a single band corresponding to a molecularweight of 90 kDa to 100 kDa on electrophoresis as described later on.The specific activity of the obtained purified enzyme sample was about2000 times that of the crude extract solution. The recovery was 12% withrespect to the amount of the enzyme obtained after the strongly basicanion exchange chromatography based on the use of HiTrap Q. Results ofthe purification are summarized in Table 1. TABLE 1 Specific Totalprotein Total activity activity mg nmol/h nmol/h/mg Yield % Crudeextract 1915 20700 11 — HiTrap Q 1092 48800 45 100  DEAE-TOYOPEARL 54033000 61 68 Superdex 200 pg 1.79 26500 14800 54 Apatite (1)* 0.51 1260024700 26 Apatite (2)* 0.20 5700 28500 12Apatite (1)*: Hydroxyapatite chromatography (1)Apatite (2)*: Hydroxyapatite chromatography (2)

EXAMPLE 2 Investigation on Characteristics of Raffinose Synthase

Characteristics of the purified raffinose synthase obtained in Example 1were investigated.

<1> Molecular Weight Measurement

(1) Gel Filtration Chromatography

An aliquot (10 μl) of the purified raffinose synthase was dispensed.This sample and a molecular weight marker (Molecular Weight Marker Kitfor Gel Filtration, produced by Pharmacia) were applied to a gelfiltration chromatography column (Superdex 200 pg, produced byPharmacia). Equilibration of the column and elution from the column wereperformed by using Buffer 6 (20 mM Tris-HCl buffer (pH 7.0), 0.1 M NaCl,5 mM DTT, 0.02% Tween 20). As a result, the molecular weight of theraffinose synthase was estimated to be about 75 kDa to 95 kDa.

(2) Polyacrylamide Gel Electrophoresis (Native PAGE)

An aliquot (10 μl) of the purified raffinose synthase was dispensed, towhich the same volume of a sample buffer (0.0625 M Tris-HCl (pH 6.8),15% glycerol, 0.001% BPB) was added to prepare an electrophoresissample. The sample (10 μl) was applied to 10% polyacrylamide gel(produced by Daiichi Chemical, Multigel 10), and electrophoresed at 40mA for about 60 minutes with 0.025 M Tris-0.192 M glycine buffer (pH8.4). After the electrophoresis, the gel was stained with Silver StainKit (produced by nacalai tesque). As a result, the molecular weight wasestimated to be about 90 kDa to 100 kDa.

(3) SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

An aliquot-(10 μl) of the purified raffinose synthase was dispensed, towhich the same volume of a sample buffer (0.0625 M Tris-HCl (pH 6.8), 2%SDS, 10% glycerol, 5% mercaptoethanol, 0.001% BPB) was added, followedby heating in a boiling water bath for 1 minute to prepare anelectrophoresis sample. The sample (10 μl) was applied to 10 to 20%gradient polyacrylamide gel (produced by Daiichi Chemical), andelectrophoresed at 40 mA for about 70 minutes with 0.025 M Tris-0.192 Mglycine buffer (pH 8.4) containing 0.1% SDS. After the electrophoresis,the gel was stained with Silver Stain Kit (produced by nacalai tesque).A result is shown in FIG. 2. As a result, the molecular weight wasestimated to be about 90 kDa to 100 kDa.

<2> Optimum Reaction Temperature

The raffinose synthase activity was measured under various temperatureconditions (28° C., 32° C., 36° C., 40° C., 44° C., 48° C., and 52° C.)in accordance with the method for measuring the raffinose synthaseactivity described above. The enzyme solution was added to therespective reaction solutions in an amount of 2 μl. FIG. 3 showsrelative activities at the respective temperatures assuming that theenzyme activity at 32° C. was 100. As a result, the raffinose synthaseexhibited the activity in a range of about 25 to 42° C., and the optimumreaction temperature was about 35 to 40° C.

<3> Optimum Reaction pH

The raffinose synthase activity was measured under various pH conditions(pH 4 to 11) in accordance with the method for measuring the raffinosesynthase activity described above. The respective reactions wereperformed by using 50 mM citrate buffer (pH 4 to 6), 50 mM potassiumphosphate buffer (pH 5.5 to 7.5), 50 mM Bis-Tris buffer (pH 6 to 7), 20mM Tris-HCl buffer (pH 7 to 8.5), and 50 mM glycine-NaOH buffer (pH 9 to11). The enzyme solution was added to the respective reaction solutionsin an amount of 2 μl. A result is shown in FIG. 4.

As a result, the raffinose synthase exhibited the activity in a range ofpH 5 to 10, and the optimum reaction pH was about 6 to 8, provided thatthe activity varied depending on the type of the buffer used for themeasurement.

<4> Investigation on Inhibitors and Metal Ions

Various enzyme inhibitors or metal ions were added to the reactionsolution of the purified raffinose synthase to give a finalconcentration of 1 mM respectively, and the raffinose synthase activitywas measured in the same manner as described above. Table 2 showsremaining activities with respect to the enzyme activity obtained whenneither inhibitor nor metal ion was added. Iodoacetamide andN-ethylmaleimide clearly inhibited the enzyme activity. The inhibitingeffect was scarcely observed for CaCl₂, CuCl₂, and MgCl₂. However,MnCl₂, ZnCl₂, and NiCl₂ exhibited the inhibiting effect. TABLE 2Inhibitor or metal ion Remaining activity (%) No addition 100Iodoacetoamide 0 N-ethylmaleimide 40 CaCl₂ 115 CuCl₂ 101 MgCl₂ 96 MnCl₂32 ZnCl₂ 42 NiCl₂ 68<5> Inhibition by Myo-Inositol

Investigation was made for inhibition by myo-inositol as the reactionproduct of the raffinose synthesis reaction. The reaction solution wasadded with myo-inositol at various concentrations, and the raffinosesynthase activity was measured. A result is shown in FIG. 5. The enzymeactivity was inhibited as the concentration of added myo-inositol wasincreased.

<6> Stable pH

The raffinose synthase fraction obtained by the anion exchangechromatography (2) described above was incubated for 4 hours at 4° C. in50 mM Bis-Tris-HCl buffer (pH 5 to 8.0, containing 0.5 mM DTT) or 20 mMTris-HCl buffer (pH 7 to 8.0, containing 0.5 mM DTT), and then theraffinose synthase activity was measured. FIG. 6 shows the enzymeactivity versus pH of the buffer used for the incubation. The raffinosesynthase activity was confirmed after the incubation under any of theincubation conditions. Especially, the enzyme was stable in a range ofpH 5 to 7.5.

<7> Stable Temperature

The raffinose synthase fraction obtained by the anion exchangechromatography (2) described above was incubated in 20 mM Tris-HClbuffer (pH 7, containing 0.5 mM DTT) for 60 minutes at 28° C., 32° C.,37° C., or 40° C., and then the raffinose synthase activity wasmeasured. As a result, the enzyme of the present invention was stable,exhibiting, in the range of 28° C. to 40° C., activities of 80% to 100%of that obtained by a control for which the incubation treatment was notperformed for comparison.

<8> Analysis of Amino Acid Sequence

The cysteine residue of the purified raffinose synthase was subjected toreducing pyridylethylation, and the reaction mixture was desalted. Anobtained sample was digested at 37° C. for 12 hours withlysylendopeptidase (Achromobacter protease 1, produced by Wako PureChemical Industries) to form peptide fragments. An obtained peptidemixture was applied to reverse phase HPLC (column: Waters μBondasphere(φ2.1×150 mm, C₁₈, 300 Å, produced by Waters (Millipore))) to separateand obtain the respective peptide fragments. 0.1% TFA (trifluoroaceticacid) was used as a solvent, and elution was performed with aconcentration gradient of acetonitrile. Amino acid sequences of threefragments selected from the obtained peptide fragments were determinedby using a protein sequencer. The determined amino acid sequences of therespective peptides are shown in SEQ ID NOs: 1 to 3 in Sequence Listing.These peptides will be thereafter referred to as Peptides 1, 2, and 3respectively in this order.

EXAMPLE 3 Preparation of DNA Coding for Raffinose Synthase

<1> Isolation of Partial Fragment of cDNA of Raffinose Synthase by Meansof PCR Method

Major veins of cucumber (22 g) were ground by a mortar with liquidnitrogen. The ground material was added to a mixture of an extractionbuffer (100 mM lithium chloride, 100 mM Tris-HCl (pH 8.0), 10 mM EDTA,and 1% SDS) and an equal amount of phenol previously heated to 80° C.,followed by agitation. After that, a mixture of phenol and an equalamount of chloroform: isoamyl alcohol (24:1) was added thereto, followedby agitation again. An obtained mixture solution was centrifuged at 4°C. at 9250×g for 15 minutes to collect a supernatant. The supernatantwas repeatedly subjected to the treatment with phenol and the treatmentwith chloroform: isoamyl alcohol to obtain a supernatant aftercentrifugation. The supernatant was added with an equal amount of 4 Mlithium chloride, followed by being stationarily left to stand at −70°C. for 1 hour.

After thawing at room temperature, the sample was treated andcentrifuged at 4° C. at 9250×g for 30 minutes to obtain a precipitate.The precipitate was washed with 2 M lithium chloride once and with 80%ethanol once. After drying, the precipitate was dissolved in 2 ml of adiethylpyrocarbonate-treated solution to give a sample of purified totalRNA. The obtained total RNA was 2.38 mg.

The all amount of the total RNA was applied to poly(A)⁺RNA purificationkit (produced by STRATAGENE CLONING SYSTEMS) based on the use of anoligo(dT) cellulose column so that poly(A)⁺RNA molecules were purifiedto obtain 42.5 μg of poly(A)⁺RNA.

Single strand cDNA's were synthesized from poly(A)⁺RNA obtained asdescribed above, by using reverse transcriptase Super Script II(produced by GIBCO BRL). In order to isolate raffinose synthase cDNAfrom an obtained cDNA mixture, amplification was performed in accordancewith the PCR method. In order to be used as primers in PCR, singlestrand oligonucleotides (SEQ ID NOs: 6 to 22) shown in FIG. 7 weresynthesized on the basis of the amino acid sequences of the peptidefragments of the raffinose synthase originating from cucumber,determined in Example 2. In the sequences of the respective primers, Rrepresents A or G, Y represents C or T, M represents A or C, Krepresents G or T, D represents G, A, or T, H represents A, T, or C, Brepresents G, T, or C, N represents G, A, T, or C, and I representsinosine (base: hypoxanthine) respectively.

A DNA fragment of about 540 base pairs was amplified when the primerswere combined and used such that the 5′-side primer was A (A1 (SEQ IDNO: 6), A2 (SEQ ID NO: 7), A3 (SEQ ID NO: 8), A4 (SEQ ID NO: 9)) and the3′-side primer was D′ (D′1 (SEQ ID NO: 21), D′2 (SEQ ID NO: 22)), or the5′-side primer was C2 (SEQ ID NO: 14) and the 3′-side primer was B′1(SEQ ID NO: 18) or B′2 (SEQ ID NO: 19). The fragment was cloned into aplasmid pCRII by using TA cloning kit (produced by INVITROGEN BV) toanalyze its nucleotide sequence. As a result, a nucleotide sequencecoding for the amino acid sequences of Peptides 1, 2 was found inwardlybetween the primer sequences at both terminals. Accordingly, it wasfound that the amplified fragment is a DNA fragment originating from theraffinose synthase gene.

In order to specify the position of the cloned PCR-amplified DNAfragment on the raffinose synthase gene, 3′-RACE was performed by usingRACE kit (3′ Ampifinder RACE Kit, produced by CLONTACH).

PCR was performed by using the cDNA mixture as a template, C (C1 (SEQ IDNO: 13), C2 (SEQ ID NO: 14)) as a 5′-side primer, and a primer havingoligo(dT) and an anchor sequence as a 3′-side primer. Further, PCR wasperformed by using an amplified fragment thus obtained as a template, D(D1 (SEQ ID NO: 15), D2 (SEQ ID NO: 16)) located inwardly from C as a5′-side primer, and an oligo(dT)-anchor primer as a 3′-side primer. As aresult, a DNA fragment of about 2400 base pairs was amplified only whenPCR was performed by using, as the template, DNA amplified with C1 (SEQID NO: 13) or C2 (SEQ ID NO: 14) and the oligo(dT)-anchor primer, andusing D2 (SEQ ID NO: 16) and the oligo(dT)-anchor primer. Further, PCRwas performed by using C (C1 (SEQ ID NO: 13), C2 (SEQ ID NO: 14)) as the5′-side primer and the oligo(dT)-anchor primer as the 3′-side primer,and then PCR was performed by using the amplified fragment thus obtainedas a template, E (SEQ ID NO: 17) as a 5′-side primer, and theoligo(dT)-anchor primer as a 3′-side primer. As a result, a DNA fragmentof about 300 base pairs was amplified in any case.

Similarly, PCR was performed by using A (A1 (SEQ ID NO: 6), A2 (SEQ IDNO: 7), A3 (SEQ ID NO: 8), or A4 (SEQ ID NO: 9)) as a 5′-side primer,and the primer having oligo(dT) and the anchor sequence as a 3′-sideprimer. Further, PCR was performed by using an amplified fragment thusobtained as a template, and using B (B1 (SEQ ID NO: 10), B2 (SEQ ID NO:11), or B3 (SEQ ID NO: 12)) located inwardly from A as a 5′-side primer,and the same oligo(dT)-anchor primer as a 3′-side primer. As a result, aDNA fragment of about 2000 base pairs was obtained when the B2 primerwas used even when any of the A primers was used. Thus the DNA fragmentamplified by using the A2 and B2 primers was cloned. As a result ofnucleotide sequence analysis, the DNA fragment included the nucleotidesequence coding for the amino acid sequence of Peptide fragment 1 usedto prepare the 5′-side primer. The DNA fragment also included, on the3′-side, the poly(A) sequence and the nucleotide sequence correspondingto Peptide fragment 3 at a position located upstream therefrom.

In view of the result of PCR described above, it was found that Peptidefragments of the raffinose synthase are arranged from the N-terminalside in an order of 2, 1, 3, and the DNA fragment of about 540 basepairs previously obtained by PCR was a portion located near to the5′-terminal on the raffinose synthase gene. In order to screen a cDNAclone containing the entire length of the raffinose synthase gene, it isdesirable that DNA to be used as a probe can detect a portion near tothe 5′-terminal side. Accordingly, the obtained DNA fragment was used asa probe to perform screening for a cDNA library.

<2> Cloning of Entire Length of Coding Region of Raffinose Synthase cDNA

At first, a cDNA library was prepared as follows. Double strand cDNA'swere synthesized from poly(A)⁺RNA (3.8 μg) obtained in the foregoingitem <1> by using Time Saver cDNA synthesis kit (produced by PharmaciaBiotech). Obtained cDNA's were incorporated into EcoRI restrictionenzyme cleavage site of λ phage vector, λMOSSlox (produced by Amersham)respectively, which were then incorporated into the phage protein byusing GigapackII Gold packaging kit (produced by STRATAGENE CLONINGSYSTEMS). Thus the cucumber cDNA library was prepared. This library hada titer of 1.46×10⁷ pfu/μg vector.

Host cells of Escherichia coli ER1647 were infected with the phagescontained in the cucumber cDNA library in an amount corresponding to1.4×10⁵ pfu, and then the cells were spread over 14 agar plates eachhaving a diameter of 90 mm to give 1.0×10⁴ pfu per one plate. The cellswere cultivated at 37° C. for about 6.5 hours. After that, phage plaquesformed on the plates were transferred to nylon membranes (Hybond-N+,produced by Amersham).

Next, the nylon membranes were treated with alkali to denaturetransferred DNA, followed by neutralization and washing. After that, thenylon membranes were treated at 80° C. for 2 hours to fix DNA on themembranes.

Positive clones were screened on the obtained nylon membrane by usingthe DNA fragment of about 540 base pairs obtained in the foregoing item<1> as a probe. The DNA fragment of about 540 base pairs was digestedwith restriction enzyme EcoRI, followed by electrophoresis to excise andpurify only the insert of about 540 base pairs. The insert was labeledwith fluorescein by using DNA labeling and detection system (Gene Imageslabeling and detection system, produced by Amersham) to be used as theprobe. The nylon membranes were subjected to prehybridization at 60° C.for 30 minutes, and then the labeled probe was added to performhybridization at 60° C. for 16 hours. An antibody (alkalinephosphatase-labeled anti-fluorescein antibody) for detecting the labeledDNA was used after being diluted 50000 times. In this screening process,candidate strains for positive clones were obtained. The obtainedcandidate strains were further subjected to repeated screening twice inthe same manner as described above to obtain a purified positive clone.

Escherichia coli BM25.8 was infected with the positive clone, and it wascultivated on a selection medium containing carbenicillin. A plasmidvector λMOSSlox-CRS containing cDNA was excised therefrom. The insertedcDNA of the plasmid had a length of about-2.5 kb. Escherichia coli JM109was transformed with the plasmid. Plasmid DNA was prepared from atransformant, which was used as a sample for analyzing the nucleotidesequence.

The nucleotide sequence of the inserted cDNA was analyzed by using TaqDyeDeoxy Terminator Cycle Sequencing Kit (produced by Perkin-Elmer) inaccordance with the conventionally known method.

As a result, a nucleotide sequence comprising 2352 base pairs as shownin SEQ ID NO: 4 in Sequence Listing was revealed. The sequence includeda portion coincident with the nucleotide sequence of the DNA probe usedby the present inventors. An amino acid sequence translated from thenucleotide sequence is shown in SEQ ID NOs: 4 and 5. The amino acidsequence included portions-coincident with Peptide 1 (amino acid numbersof 215 to 244 in SEQ ID NO: 5), Peptide 2 (amino acid numbers of 61 to79 in SEQ ID NO: 5), and Peptide 3 (amino acid numbers of 756 to 769 inSEQ ID NO: 5) of the raffinose synthase originating from cucumberobtained by the present inventors. Thus it was confirmed that the aminoacid sequence codes for the raffinose synthase.

The transformant, designated as AJ13263, of Escherichia coli JM109,which harbors the plasmid pMossloxCRS containing DNA coding for theraffinose synthase obtained as described above, has been internationallydeposited on the basis of the Budapest Treaty since Nov. 19, 1996 inNational Institute of Bioscience and Human Technology of Agency ofIndustrial Science and Technology of Ministry of International Trade andIndustry (postal code: 305, 1-3 Higashi-Icchome, Tsukuba-shi,Ibaraki-ken, Japan), and awarded a deposition number of FERM BP-5748.

EXAMPLE 4 Chimeric Gene and Transformed Plant Containing DNA Coding forRaffinose Synthase

<1> Construction of Plasmid Containing Chimeric Gene

The DNA fragment coding for the raffinose synthase was introduced intoArabidopsis thaliana by using LBA4404 as Agrobacterium and pBI121(produced by CLONTECH) as a binary vector. pBI121 is a plasmidoriginating from pBIN19, which comprises nopaline synthase gene promoterconnected to neomycin phosphotransferase structural gene (NPTII),nopaline synthase gene terminator (Nos-ter), CaMV 35S promoter, GUS(β-glucuronidase) gene, and Nos-ter, and which has sequences forenabling transposition to plant, on both sides thereof. A SmaI site islocated downstream from CaMV 35S promoter. An insert inserted into thissite is expressed under the regulation of the promoter.

A fragment of the raffinose synthase gene obtained in Example 3 wasinserted into the binary vector pBI121. The raffinose synthase gene wasdigested with DraI to prepare, by means of agarose gel electrophoresis,a DNA fragment containing 30th to 1342th nucleotides in SEQ ID NO: 4 inSequence Listing. This fragment was ligated into the SmaI site ofpBI121. Escherichia coli HB101 was transformed with the ligationreaction solution to obtain transformant strains from which recombinantplasmids were prepared. Two recombinant plasmids, in which the raffinosesynthase DNA fragment was reversely connected to CaMV 35S promoter(antisense), and the raffinose synthase DNA fragment was connected toCaMV 35S promoter in the positive direction (sense), were selected fromthe obtained recombinant plasmids. The two recombinant plasmids weredesignated as pBIRS1 and pBIRS9 respectively.

Each of the plasmids obtained as described above was introduced intoAgrobacterium LBA4404 by means of triparental mating.

Arabidopsis thaliana was transformed as follows. Seeds of Arabidopsisthaliana was subjected to a treatment for water absorption. After that,they were sterilized by treating them with 80% ethanol containing 1%Tween 20 for 5 minutes, and treating them with 10% sodium hypochloritesolution also containing 1% Tween 20 for 10 minutes, followed by washingfive times with sterilized water. The seeds were suspended in 1% lowmelting point agarose, and they were spread over an MS medium (MS basicmedium (Murashige and Skoog, Physiologia Plantrum, 15, 473-497 (1962)),B5 vitamin, 10 g/L sucrose, 0.5 g/L MES, pH 5.8). The seeds werecultivated at 22° C. for 1 week in a culture room to give a cyclecomprising light irradiation for 16 hours and darkness for 8 hours.Plants with unfolded seed leaves were subjected to setting with rockwool. Cultivation was continued under the same condition. After about 3weeks, decapitation was performed when the plants caused bolting to haveheights of stems of several cm's. The plants were allowed to grow untila state in which first flowers bloom on elongated branches 1 week afterthe decapitation.

Agrobacterium harboring the introduced recombinant plasmid containingthe raffinose synthase gene was precultivated in 2 ml of LB medium. Anobtained culture was inoculated into LB medium containing 50 mg/Lkanamycin and 25 mg/L streptomycin, followed by cultivation at 28° C.for about 1 day. Bacterial cells were collected at room temperature, andthey were suspended in a suspension medium for infiltration (½ MS salt,½ Gamborg B5 vitamin, 5% sucrose, 0.5 g/L MES, pH 5.7 (KOH), to which,immediately before the use, benzylaminopurine was added to give a finalconcentration of 0.044 μM, or Silwet L77 was added in an amount of 200μl per 1 L (final concentration: 0.02%)) so that OD₆₀₀ of an obtainedbacterial suspension was 0.8.

Flowers in bloom and fructification were removed from the plants to besubjected to infiltration. The rock wool was inverted upside down, andflowers which were not in fructification were immersed in the suspensionof Agrobacterium, followed by being placed in a desiccator so that thepressure was reduced to be 40 mmHG for 15 minutes. Seeds were harvestedafter 2 to 4 weeks. The harvested seeds were stored in a desiccator.

Next, transformants were selected on a selection medium. The seeds weresterilized in the same manner as described above, and they werecultivated on a selection medium (MS salt, Gamborg B5 vitamin, 1%sucrose, 0.5 g/L MES, pH 5.8, 0.8% agar, to which antibiotics forselection, i.e., carbenicillin (final concentration: 100 mg/L) andkanamycin (final concentration: 50 mg/L) were added after autoclaving))at 22° C. to select resistant plants. The resistant plants weretransferred to a fresh medium, and they were allowed to grow until trueleaves unfolded. Seeds were harvested from the obtained plants.Selection was repeated in the same manner as described above, and thusT3 seeds were obtained. The T3 seeds were measured for the raffinosecontent in accordance with the method described above. Results are shownin Table 4. TABLE 4 Plant Raffinose content (mg/g) Wild type 0.2Transformant (pBIRS1) 0.0 Transformant (pBIRS9) 0.0

INDUSTRIAL APPLICABILITY

The present invention provides the purified raffinose synthase, theraffinose synthase gene, the chimeric gene comprising the raffinosesynthase gene and the regulatory region expressible in plants, and theplant introduced with the chimeric gene.

Raffinose can be efficiently synthesized from sucrose and galactinol byusing the raffinose synthase of the present invention. The content ofthe raffinose family oligosaccharides in plants can be changed byutilizing the raffinose synthase gene or the chimeric gene of thepresent invention.

1-12. Canceled.
 13. A method of producing a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, comprising screening a cDNA library prepared from a plant by hybridization using a probe whose sequence is a portion of the nucleotide sequence of SEQ ID NO: 4, to select the DNA.
 14. A method of producing a vector comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, comprising producing the DNA by the method of claim 13, and inserting the DNA to a vector.
 15. A method of producing a transformed plant, comprising producing a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol by the method of claim 13, and transforming a plant with the DNA.
 16. A method according to claim 15, wherein the plant is soybean, rapeseed, cotton, sugar beet, or sugar cane.
 17. A method of producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, comprising producing the DNA by the method of claim 13, and operably linking the DNA to the transcription regulatory region expressible in plant cells.
 18. A method of producing a transformed plant, comprising producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, by the method of claim 17, and transforming a plant with the chimeric gene.
 19. The method according to claim 18, wherein the plant is soybean, rapeseed, cotton, sugar beet, or sugar cane.
 20. A method of modifying the content of raffinose family oligosaccharides in a plant, comprising producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, by the method of claim 17, and transforming the plant with the chimeric gene, thereby changing the content of raffinose family oligosaccharides in the plant.
 21. A method of producing a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, comprising subjecting a DNA coding for a protein comprising SEQ ID NO: 5 to a mutagenesis and screening for a mutant DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol.
 22. A method of producing a vector comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, comprising producing the DNA by the method of claim 21, and inserting the DNA to a vector.
 23. A method of producing a transformed plant, comprising producing a DNA which odes for a protein having an activity to produce raffinose from sucrose and galactinol by the method of claim 21, and transforming a plant with the DNA.
 24. The method according to claim 23, wherein the plant is soybean, rapeseed, cotton, sugar beet, or sugar cane.
 25. A method of producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, comprising producing the DNA by the method of claim 21, and operably linking the DNA to the transcription regulatory region expressible in the plant cells.
 26. A method of producing a transformed plant, comprising producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, by the method of claim 25, and transforming a plant with the chimeric gene.
 27. The method according to claim 26, wherein the plant is soybean, rapeseed, cotton, sugar beet, or sugar cane.
 28. A method of modifying the content of raffinose family oligosaccharides in a plant, which method comprising producing a chimeric gene comprising a DNA which codes for a protein having an activity to produce raffinose from sucrose and galactinol, operably linked to a transcription regulatory region expressible in plant cells, by the method of claim 25, and transforming the plant with the chimeric gene, thereby changing the content of raffinose family oligosaccharides in the plant. 